Kell Blood Group System (KEL) ISBT 006

Transcription

1 Kell, Duffy, Kidd Objectives: Kell, Kidd and Duffy Kell and Duffy in 30 minutes you ve got to be Kidd-ing! (Ok, we ll talk about Kidd, too) Jessica Drouillard, SBB(ASCP) CM Heartland Blood Centers, part of Versiti Aurora, IL For each blood group discussed, the learner will: State blood group antigen frequencies among the general population and within specific ethnic groups Appreciate the genetics and biochemistry Discuss implications of null phenotypes List the characteristics of antibodies directed against each blood group Discuss the use of chemicals in antibody identification Identify diseases related to blood groups Inheritance XK gene on X chromosome Xk protein Kx antigen Kell Blood Group System (KEL) ISBT 006 KEL gene on Chromosome 7 Kell glycoprotein Kell antigens KEL and XK genes interact to form normal Kell antigen expression Expression Normal Kell Expression Kell antigens -K, k -Kp a, Kp b -Js a, Js b No Kx Weak Kell antigens Kell antigens -K, k -Kp a, Kp b -Js a, Js b Antigen ISBT Whites (%) Blacks (%) K KEL1 9 2 k KEL Kp a KEL3 2 Rare Kp b KEL Kx Kx No Kell Ku- Km- Kx Ku KEL Js a KEL Js b KEL

2 Kell, Duffy, Kidd Ko phenotype McLeod Phenotype -SNP of KEL*02 -Homozygous -No Kell glycoproteins -Null phenotype -Cells type Kx+ s Ku- Km- -Can form anti-ku and anti-km Ko phenotype Kx Normal Kx No Kell Ku- Km- Kell antigens -K, k -Kp a, Kp b -Js a, Js b No XK gene No Kx No Km Can form anti-kx and Km Other Kell typings weak Ku K, k, Kpa, Kpb, Jsa, Jsb McLeod phenotype No Kx Normal Kx Weak Kell antigens Kell antigens -K, k -Kp a, Kp b -Js a, Js b McLeod Syndrome Antibodies X linked, occurs almost exclusively in males Some patients also have X-linked Chronic Granulomatous Disease (CGD) Range of neurological and muscular defects Muscle wasting, reduction in deep tendon reflexes Hematologic abnormalities Decreased RBC survival, acanthocytosis, reticulocytosis, reduced serum haptoglobin, increased bilirubin, compensated anemia Difficult to find blood for transfusion Primarily IgG, do not bind complement Usually immune stimulated Clinically significant (HTR and HDFN) Ko phenotype Phenotype Antibody Compatible blood Kx McLeod phenotype (no CGD) No Kx McLeod phenotype with CGD No Kx No Kell Ku- Km- Weak Kell antigens Weak Kell antigens Anti-Ku Anti-Km Anti-Km Anti-Kx Anti-Kx, -Km Ko Ko and McLeod Ko and McLeod McLeod McLeod Weakened Kell Antigen Expression K mod K mod phenotypes Kp a in cis k, Kp a, Js b Possible Kell haplotypes k, Kp b, Js b K, Kp b, Js b k, Kp Gerbich null types a, Js b k, Kp Ge:2,3,4 is normal b, Js a Ge:-2,3,4 = Yus - Kell normal Ge:-2,-3, 4 = Gerbich Kell expression weakened Ge:-2,-3,-4 = Leach (true null) Kell expression depressed Arises from SNP at KEL*02 Weakened expression of Kell glycoproteins Often need adsorption/elution studies to detect Strong Kx expression Some may make anti-kulike antibody that reacts with all cells but other K mod Kx K mod Normal Kell antigens -K, k -Kp a, Kp b -Js a, Js b 38

3 Kell, Duffy, Kidd Inheritance Duffy Blood Group System ISBT 008 FY*01 and FY*02 genes on Chromosome 1 Syntenic to Rh Produce Duffy glycoproteins: Fy3, Fy5, Fy6, and Fy a /Fy b Duffy Antigen Receptor for Chemokines Binds cytokines, especially IL-8 Role in inflammation Also receptor for malaria Plasmodium vivax Plasmodium knowlesi Expression Duffy Phenotypes Well developed at birth Destroyed by enzymes and ZZAP Antigens found on RBCs and other tissues Endothelial cells, brain, colon, lung, spleen, kidney, etc Antigens reported to weaken when stored Fy6 Fy a /Fy b Fy3 RBC Phenotype Whites (%) Blacks (%) Asians (%) Fy(a+b-) Fy(a+b+) Fy(a-b+) <1 Fy(a-b-) Fy Fy Fy x Fy(a-b-) type Duffy genotypes Rare in whites, but usually true null Duffy antigens not expressed on RBCs or tissues Can make anti-fy3 Most frequently found in blacks Arises from SNP mutation in GATA-1 erythroid promoter region of FY*01 (Fy a ) or FY*02 (Fy b ) more common Duffy antigens not expressed on RBCs, but are expressed on tissues Genotype RBC Phenotype Fya Fyb Fy3 Comments FY*A / FY*A Normal, homozygous Fya expression FY*A / FY*B Normal, heterozygous Fya/Fyb expression FY*B / FY*B Normal, homozygous Fyb expression FY*A / FY*B_GATA Fya expressed on RBC and tissues Fyb expressed on tissues (not RBC) Should not make anti-fyb FY*B / FY*B_GATA Fyb expressed on RBC and tissues Can make anti-fya FY*B_GATA / FY*B_GATA Fy(a-b-) phenotype Fyb expressed on tissues (not RBC) Can make anti-fya Should not make anti-fyb Fymod / Fymod w+ w+ w+ Weak Fy6 antigen expression 39

4 Kell, Duffy, Kidd Duffy antibodies Usually IgG, do not bind complement well Show dosage Anti-Fy a is 20 times more common than anti-fy b Clinically significant (HTR and HDFN, usually mild) Antigen Fy a / Fy b Fy3 Fy5 Fy6 Reaction with enzymes Destroyed Resistant Resistant Destroyed Other Duffy antibodies Anti-Fy3 Found only in Fy(a-b-) individuals Reacts like an inseparable anti-fya, -Fyb, but enzymes will not eliminate reactivity Anti-Fy5 Found only in Fy(a-b-) individuals Reacts with all cells except Fy(a-b-) Rh null cells, regardless of Fy a /Fy b typing D-- cells have weak Fy5 expression Anti-Fy6 Murine antibody not found in humans Inheritance Kidd Blood Group System ISBT 009 JK*01 and JK*02 found on Chromosome 18 Human Urea Transporter 11 (HUT11) Allows uptake of urea Prevents RBC shrinkage in hypertonic environment of renal medulla Jk3 antigen absent/weak in Jk(a-b-) individuals Dominant inhibitor gene In(Jk) Reported in Japanese families No Jk a or Jk b expression Weak Jk3 expression Homozygous for Jk gene true JK null (No Jk a, Jk b or Jk3) Polynesians and Finns Expression Well developed at birth Antigens cluster on RBC surface Transmembrane protein (like Rh) Not destroyed by DTT/AET or enzymes Enhanced by enzymes Kidd Phenotypes Phenotype Whites (%) Blacks (%) Jk(a+b-), Jk Jk(a+b+), Jk Jk(a-b+), Jk Jk(a-b-), Jk3- Polynesians, Finns Jk(a-b-), Jk3+ (weak) Japanese Poor immunogens 40

5 Kell, Duffy, Kidd Jk(a-b-) Phenotype When caused by dominant inhibitor gene Need only 1 copy of In(Jk) gene Cells type Jk(a-b-) and Jk3-, but antigens may be detected by adsorption/elution studies Since trace amounts of antigens are present, cannot make anti-jk3 When caused by inheritance of JK gene Need 2 copies of JK gene (must be homozygous) Cells type Jk(a-b-) and Jk3- Kidd antigens completely absent Can make anti-jk3 2M Urea Lysis Test Cheap, effective screening test to identify Jk(a-b-) individuals Cells with normal Kidd antigen expression have normal HUT11, will swell and lyse Jk(a-b-) cells resist lysis by 2M urea, but shrink and shrivel 2M Urea Lysis Test Kidd Antibodies Disappear quickly from circulation and don t store well Associated with DHTR Primarily IgG1, IgG3 Can activate compliment Antigens clustered together on RBC surface Clinically significant can cause HTR and rarely HDFN Anti-Jk3 made by true Jk(a-b-) individuals Looks like inseparable anti-jk a and anti-jk b Confirm with adsorption/elution studies Can you guess each donor s likely ethnicity? Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Review Questions

6 Kell, Duffy, Kidd Can you guess each donor s likely ethnicity? Answers Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb , 5 African American / Black 2, 3, 4 White 6 Japanese, Finnish, Polynesian (?) 7 Unknown Which cell likely has HOMOZYGOUS expression of the Fya antigen? Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Which cell likely has HOMOZYGOUS expression of the Fya antigen? ANSWER Cell K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb What is the most likely cause of the reaction pattern observed below? Cell K k Kpa Kpb Jsa Jsb Anti-k (weak) Anti-Jsb (weak) A Anti-k antisera is expired B Cell 4 is from a patient with McLeod phenotype C Cell 4 is from a Kx patient D Cell 4 is from a K mod patient E Kp a effect F Cell 4 is from a Ge: -2, -3, -4 patient What is the most likely cause of the reaction pattern observed below? ANSWER Cell K k Kpa Kpb Jsa Jsb Anti-k (weak) Anti-Jsb (weak) A Anti-k antisera is expired B Cell 4 is from a patient with McLeod phenotype C Cell 4 is from a Kx patient D Cell 4 is from a K mod patient E Kp a effect F Cell 4 is from a Ge: -2, -3, -4 patient 42

7 MNS and Others This presentation highlights selected systems; it is not intended to be a comprehensive review MNS and Other Blood Groups Cindy Piefer, MT(ASCP)SBB Manager, Immunohematology Reference Laboratory Objectives Discuss the antigens, gene location, protein, nomenclature, and phenotype distribution Describe the serological characteristics of antibodies to antigens in these systems Discuss investigational techniques for identifying the antibodies Describe the clinical significance of antibodies in transfusion and in HDFN MNS Blood Group System (ISBT 002) Chromosome 4 Genes GYPA, GYPB Glycophorin B (GPB) Gene products Glycophorin A (GPA) & Glycophorin function > sialic acid contributes to the negative charge on red cells GPA and GPB are type 1 transmembrane sialoglycoproteins, cleaved by proteolytic enzymes GYPE adjacent to GYPB, no RBC membrane product, believed to cause the variant/hybrid alleles Gylcophorin A and B Glycophorin Comparison Glycophorin A 1 million copies per RBC 131 amino acids M: Ser-Ser-Thr-Thr-Gly N: Leu-Ser-Thr-Thr-Glu Glycophorin B 200,000 copies per RBC 72 amino acids S: 48 Methionine(previously 29) s: 48 Threonine(previously 29) N first 26 aa same as N GPA Not cleaved by trypsin 43

8 MNS and Others Null Phenotypes En(a-) (MNS28) Deletion of GYPA and/or GYPB results in the silencing of the genes; no gene products are made Deficient Glycophorin Phenotype Deletion of exons En(a-) No GPA M-N-En(a-) GYPA (exon 2-7) and GYPB (exon 1) U- No GPB S-s-U- GYPB (exon 2-6) and GYPE (exon 1) M k No GPA or GPB M k M k M-N-En(a-) S-s-U- GYPA (exon 2-7) GYPB (exon 1-6) GYPE (exon 1) En means Ag carried on the envelope of RBC, high-prevalence antigen En(a-) cells lack GPA or have variant form GPA is closely associated with Band 3, required for expression of Wr b Type as Wr(a-b-) Enzyme testing can determine antibody specificity Resistant to DTT and Chymotrypsin No to severe HTR and HDFN U (MNS5) and U variants High prevalence antigen, 99% of AA are U+ Result from the absence of GPB (S-s-) or an altered (hybrid) form of GPB (He, Dantu, SAT and St a ) Dantu+, S- s+weak 49% of S-s- are U var 37% of these are He+ Ficin resistant Molecular testing better for detecting U var Low Prevalence Antigens Hybrid gene: crossing over between GPA and GPB give rise to rare, low-prevalence variant alleles Mur is low, but more common in Southeast Asia Up to 90% in certain regions of Taiwan Anti-Mur can cause severe HTRs and HDFN Anti-Mur most common after anti-a and anti-b Mur+ red cell important on screening cells in SE Asia Others: M g MN allele; previously used in paternity MNS Antibodies Anti-M more common, anti-n rare Show dosage Anti-M enhanced at ph <65/acidified serum Anti-N reagent may be Vicia graminea lectin Anti-N associated with dialysis equipment formaldehyde treatment Most anti-m and -N are not clinically significant If PW+ at 37C or IgG: give antigen neg and do IAT XM Anti-S, -s, -U: usually IgG, AHTRs/DHTRs, HDFN Lutheran Blood Group System (ISBT 005) Chromosome 19; linked to Se 24 antigens; four antithetical pairs: Lu a (LU1)/Lu b (LU2) Lu6/Lu9 Lu8/Lu14 Au a (LU18)/Au b (LU19) Sensitive to trypsin, AET, DTT Resistant to ficin and papain 44

9 MNS and Others LU Phenotypes The Nulls Reaction with anti-lu a Reaction with anti-lu b Phenotype Incidence (%) Most of the red cells are normal, but may be acanthocytic May be due to Lu gp binding to spectrin + 0 Lu(a+b-) Lu(a+b+) Lu(a-b+) Lu(a-b-)* RARE Three Types Recessive Silent allele at the Lu locus Dominant Suppressor gene at a separate locus X-linked Suppressor gene on the X chromosome * Lu(a-b-): Three different types Recessive Lu(a-b-) Recessive silent allele; amorphic Lu gene inherited from both parents LuLu cells are Lu(a-b-) Only form that can make anti-lu3 and/or -Lua, -Lub Dominant Lu(a-b-) Dominant inhibitor In(Lu), most common (1 in 3000 or 003%) Cells are Lu(a-b-) but can be detected by adsorption - elution No antibody production Decreased expression of P1, i, AnWj, In, Knops, Cost and MER2 antigens X-linked Lu(a-b-) aka Lu mod X-linked gene daughters are carriers Daughters will have normal expression if father s expression is normal (XS2/X vs XS2/XS2) Sons are affected (XS2/Y) No antibody production Lutheran Antibodies Loose or stringy mixed-field agglutination Naturally occurring, IgM and IgA Most are immune: IgG Anti-Lua and anti-lub have caused mild DHTRs; anti-lu8 AHTRs Do not cause HDFN; antigens not fully developed at birth AET/DTT sensitive (Lu ag located in the disulfide-bonded domains) Ficin resistant Capillary testing: pine tree-like appearance 45

10 MNS and Others Diego Blood Group System (ISBT 010) 22 Antigens Assigned to Diego DI gene located on Band 3 or Anion Exchanger 1 (AE1) Maintains the structural integrity of the red cell Allows anion (HCO3- and Cl-) exchange across red cell membrane High Prevalence (only 3 Ag) Low Prevalence (19 Ag s) Adapted from Reid ME, Lomas-Francis C The Blood Group Antigen Facts Book, 2 nd ed 2004 Di b Wr b DISK Di a Wr a Wu 16 others Di a is low in Caucasians and Blacks, but higher in: South American Indians ~36% Japanese 12%, US Mexicans 10%, Chinese 5% Wr(b-) lacks GPA = En a negative Resistant to enzymes and DTT/AET Diego System Antibodies YT Blood Group System (ISBT 011) Anti-Di a or Anti-Di b Anti-Wr a Anti-Wr b IgG1 and IgG3 Anti-Di a : DHTR and HDFN Anti-Di b rare HTR; can be an autoantibody Anti-Di b demonstrates dosage RT (IgM), IAT (IgG1) Common antibody Naturally occurring in 1-2% of donors Severe HDFN and HTRs Common in AIHA Alloantibody: rare Autoab: common and may be implicated in AIHA Cases of acute & delayed HTRs HDFN DAT+ not clinical finding Two antigens on acetylcholinesterase (AChE) Yt a (high prevalence) and Yt b (8%) Chemicals: Ficin variable DTT and chymotrypsin sensitive Trypsin resistant Anti-Yt a ; questionable clinical significance (IgG1 & IgG4) No HDFN XG Blood Group System (ISBT 012) Xg Antibodies & Use Gene on X chromosome Two antigens: Xg a 66%males and 89% females CD99 (high prevalence) Anti-Xga IgG Some are naturally occurring No HTRs or HDFN (weak expression on cord RBCs) Chemicals: Ficin, trypsin, and chymotrypsin sensitive DTT resistant Genetic uses Disproved Lyon hypothesis of one X chromosome being inactivated early in embryonic life 46

11 MNS and Others Colton Blood Group System (ISBT 015) Co a - High-prevalence antigen Co b - Antithetical antigen, prevalence of about 8% in Whites, lower in other ethnic groups Co(a-b-) null phenotype makes anti-co3 Resistant to chemicals (ficin and DTT) Anti-Co a /Co b have caused HTRs and HDFN Anti-Co b occurs in sera that contain other antibodies Gerbich Blood Group System (ISBT 020) 12 antigens: 7 high prevalence and 5 low prevalence antigens Carried on glycophorin C and D (GPC, GPD) Interact directly with protein band 41 and p55, Contributes to RBC membrane stability 41-deficient RBCs can be associated with elliptocytosis GE:2,3,4 in >99% population RBC receptor for Influenza A and Influenza B Adapted from Reid ME, Lomas-Francis C The Blood Group Antigen Facts Book, 2 nd ed 2004 Phenotype Name Nucleotide Change GE: -2, 3, 4 Yus Gerbich Phenotypes Deletion exon 2 altered GPC Ethnicity Occurrence Hispanic, Israeli, Mediterranean (rare) Can make Antibody Anti-Ge2 Kell and Vel typing Normal Gerbich Antibodies Mostly IgG; may have IgM component Do not bind complement Generally not considered clinically significant, but clinically significant antibodies include Anti-Ge2 and Anti-Ge3 in HDFN Autoanti-Ge2, -Ge3 reported in AIHA cases Ficin treatment: differentiates anti-ge3 GE: -2,-3, 4 Gerbich Deletion exon 3 altered GPC GE: -2,-3,-4 Leach Deletion exon 3 & 4 Melanesians (50%) Rare Anti-Ge2 or Anti-Ge3 Anti-Ge2 or Anti-Ge3 or Anti-Ge4 Weak Weak Antigen Ge2 Ge3 Ge4 Destroyed by Ficin and/or Papain Yes NO Yes Cromer Blood Group System (ISBT 021) 18 antigens on complement-regulatory glycoprotein (DAF, decay acceleratory factor, or CD55) DAF deficiency is associated with PNH 15 high prevalence antigens 3 low prevalence antigens: Tc b, Tc c, Wes a Antithetical pairs: Tc a /Tc b /Tc c WES a /WES b Null phenotype = Inab phenotype can make anti-ifc Cromer Antigens and Antibodies Antigens present in serum/plasma, urine, platelets, WBC and placental tissues Depressed during pregnancy, and poorly expressed on cord cells Chemicals: Ficin resistant and weakened with DTT None to moderate HTR Does not cause HDFN DAF on surface of trophoblasts in the placenta 47

12 MNS and Others Indian Blood Group System (ISBT 023) Indian glycoprotein CD44 Two Antigens: In a (low), In b (high) Sensitive to ficin, DTT, trypsin, chymotrypsin Weak on cord cells, pregnant woman and In(Lu) RBCs Antibodies: HTR: anti-in a none; anti-in b none to severe/delayed and hemolytic HDFN: no, DAT may be positive VEL Blood Group System (ISBT 034) Vel- RBCs found in 1:4000 people and 1:1700 Norwegians and Swedes Chemicals: Ficin, trypsin, chymotrypsin resistant (enhanced) DTT: variable/resistant Anti-Vel: IgM and IgG, bind complement, some hemolytic HTR: mild to severe/hemolytic and HDFN: rare May be an autoantibody The effect of enzymes and DTT on antigens Ficin/Papain DTT Possible Specificty Negative Positive M,N,S,s*; Ge2, Ge4; Xg a Negative Negative Indian Positive Weak Cromer, Lutheran Thank you! I appreciate all the help from the various blood bank leaders who have studied these systems and determined the information I have shared with you Any questions? Variable Negative Yt a Positive Positive Diego; Colton; Ge3; Vel *s variable expression with ficin/papain 48

13 AIHA Autoimmune Hemolytic Anemias Sue Johnson, MSTM, MT(ASCP)SBB Director, Clinical Education BloodCenter of Wisconsin Milwaukee, WI Objectives Describe the serologic findings that characterize WAIHA and cold agglutinin syndrome Discuss the laboratory investigation and methods used to evaluate patients with autoimmune hemolytic anemia Discuss the role of blood transfusion in autoimmune disorders, problems encountered in obtaining "compatible blood" and approaches to transfusion Describe serologic findings in the following unusual cases of autoimmune hemolytic anemia: DAT negative WAIHA Mixed Type associated with both warm and cold-reactive autoantibodies IgM warm-reactive autoantibodies Objectives Describe initial serologic results observed in a patient with druginduced immune hemolytic anemia Discuss methods used to detect drug-dependent red cell antibodies in serum and eluates Drug treated red blood cells In the presence of drug Drug metabolites (urine and serum) Describe proposed immunological "mechanisms" of DIIHA & common drugs associated with each category Hapten-dependent antibody (Drug adsorption) - drug binds firmly to RBC membrane Drug-dependent antibody binds to untreated RBCs in presence of drug Nonimmunologic protein adsorption Drug-independent autoantibody autoantibody induced by drug Anti-C3 Strength of Reaction? Acute or DIIHA PCH Delayed HTR *Serological results must be correlated with Acute or clinical findings Delayed HTR Positive Polyspecific DAT Perform DAT with Monospecific Reagents and Controls What is Both Anti-IgG and C3 Positive? Strength of Reaction? Anti-IgG CAD Acute or DIIHA WAIHA Delayed Strength of HTR Reaction? Passively ABO Rh Other DIIHA WAIHA Acquired HDFN HDFN HDFN Antibody AIHA Serologic Types Barcellini et al Blood 2014 Nov 6;124(19): Serologic Type % Warm, DAT IgG only 43 Warm, DAT - IgG & C3 17 Cold, DAT - C3 27 Mixed, DAT - IgG & C3, Warm & 8 Cold Autoantibody Atypical 5 Total Patients Courtesy of C Feldman & J O Connor 49

14 AIHA Warm Autoimmune Hemolytic Anemia Incidence and Cause 1º - idiopathic 2 º - lymphoma, SLE, carcinoma Most common type of immune hemolysis IgG antibody reactive at 37ºC Warm Autoantibody in Plasma Warm Autoantibody Coating RBCs Warm Autoantibody in Plasma (1+) Warm Autoantibody in Plasma (3+) Antibody Identification Saline or LISS D C c E e K Fy a Fy b Jk a Jk b S s IAT Auto 4 50

15 AIHA Warm Autoantibody Common Specificities Rh-related Broad: Negative with Rh null cells Single: -c, -e, -D, -C, -E, -f Relative: c-like, e-like Uncommon Specificities M, N, S, U, En a FS, En a FR, Wr b K, Kp b, K13 LW a, LW ab, Jk a, Jk b, Jk3 A, B, I T Ge, Vel, AnWj, Sc1, Sc3, R x Relative Specificity: -e-like D C c E e K Fy a Fy b Jk a Jk b S s IAT Auto 4 Antibody Identification Saline D C c E e K Fy a Fy b Jk a Jk b S s IAT IAT Auto 4 Autologous Adsorption Prepare DAT Negative RBCs ZZAP Ficin or Papain 2-ME or DTT WARM TM Enzymes Ficin or Papain ZZAP Treat Patient RBCs After ZZAP Treating 51

16 AIHA Autologous Adsorption Autologous Adsorption ZZAP RBCs Pt Serum + ZZAP RBCs Mix & Incubate at 37C Centrifuge Pt Serum + ZZAP RBCs Adsorbed Serum = Anti-Jk a = WAA = Anti-Jk a = WAA Allogeneic Adsorption Other Methods Gluteraldehyde -Treated RBCs Stroma PEG Other Considerations Perform cell separation & phenotype or genotype Select phenotype matched RBCs Treat RBCs with ZZAP or enzymes Allogeneic Adsorption Patient s Phenotype is Unknown RBC Phenotype Antibodies Remaining R 1 R 1 ; Jk(a-) -c, -E, -Jk a R 2 R 2 ; Jk(b-); S- -e, -C, -Jk b, -S rr; K-; s- -D, -C, -E, -s Allogeneic Adsorption Allogeneic Adsorption After 37C Incubation Pt Serum + R 1 R 1, Jk(a-) RBCs Pt Serum + R 2 R 2, Jk(a+) RBCs Pt Serum + rr, Jk(a-) RBCs Pt Serum + R 1 R 1, Jk(a-) RBCs Pt Serum + R 2 R 2, Jk(a+) RBCs Pt Serum + rr, Jk(a-) RBCs = Anti-Jk a = Anti-Jk a = WAA = Jk(a+) RBCs = Jk(a+) RBCs 52

17 AIHA Testing Adsorbed Serum/Plasma Positive Results Adsorption not complete Reactivity is weakened but consistent with RBCs tested Perform additional adsorptions Antigen destroyed by enzymes or ZZAP No change in reactivity post adsorption Test raw serum with enzymes or ZZAP treated RBCs Perform adsorptions with untreated RBCs Reactivity due to alloantibody Some positive, some negative results Test adsorbed serum with DAT negative RBCs Test adsorbed serum with phenotypically similar RBCs Identify alloantibody R 1 R 1, Jk(a-) IAT Alloadsorbed Serum Saline Tube Serum Adsorbed with R 2 R 2, Jk(a+) IAT rr, Jk(a-) IAT I Jk(a+) II Jk(a-) III Jk(a+) R 1 R 1, Jk(a-) IAT R 2 R 2, Jk(a+) IAT rr, Jk(a-) IAT I Jk(a+) II Jk(a-) III Jk(a+) Serum Adsorbed with R 1 R 1, Jk(a-) IAT R 2 R 2, Jk(a+) IAT rr, Jk(a-) IAT I Jk(a+) II Jk(a-) III Jk(a+) Antigen on adsorbing RBCs denatured Adsorption not complete No adsorption Serum Adsorbed with Underlying alloantibody ELUATE Last Eluate Wash D C c E e K Fy a Fy b Jk a Jk b S s IAT IAT WAIHA vs DIIHA Eluate WAA strongly positive DDA is negative or weak Serum WAA persists DDA disappears within days if drug is discontinued Auto NT Management of WAIHA Steroids Rituximab Immunosuppressive drugs - Cytoxan Intravenous Immune Globulin (IVIG) Plasmapheresis Others, Cyclosporine Avoid transfusion unless life-threatening hemolysis! Unusual AIHAs ~13% of AIHAs* are Unusual Types *Barcellini et al Blood 2014 Nov 6;124(19): ~ 8% Mixed Type AIHA* DAT positive with both IgG & C3 Warm & Cold Autoantibody present DAT-Negative AIHA IgA warm autoantibody-induced AIHA IgM warm autoantibody-induced AIHA IgM warm-reactive autoantibody IS 37C IAT I II

18 AIHA WAIHA Associated With a Negative DAT 1-5% of patients with signs & symptoms of hemolysis Low affinity IgG autoantibodies Small amount of bound IgG IgM and IgA autoantibodies Enhanced DAT Methods Cold Saline Wash Cold LISS Wash Direct PEG Direct Polybrene ELAT Flow Cytometry Cold-Hemagglutinin Disease (CHD) Acute Lymphoproliferative disorders Mycoplasma pneumonia infection Infectious mononucleosis Chronic More severe symptoms Elderly, during cold weather Hemolysis mildmoderate Serology DAT shows complement only Antibody characteristics Increased thermal amplitude IgM antibody Reacts up to 37ºC in albumin Titer > 1000 at 4ºC Cold-Reactive Autoantibodies Phase & Strength of Reactivity IS 37C IAT I II Antibody specificity Most commonly anti-i Occasionally anti-i associated with infectious mononucleosis Clinical Manifestations of CAD Mild chronic anemia Occasional jaundice and pallor Some patients have increased episodes of hemolysis associated with hemoglobinuria and hemoglobinemia upon cold exposure Hemolysis usually self-limited if associated with mycoplasma or other viral infection Management of CAD Avoid cold Plasmapheresis - improvements only temporary Steroids and splenectomy of little benefit IVIG of little benefit Drug-Induced Immune Hemolytic Anemia DAT - Reactivity depends on time of testing compared to presentation Polyspecific AHG 3-4+ Anti-IgG 3-4+ Anti-C Control 0 Eluate Rapid Acid Most are negative Few are disproportionately weaker as compared to strength of DAT 54

19 AIHA DIIHA Serum Reactivity Reactivity depends on time of testing compared to presentation Saline IAT - some positive PEG or Ficin IAT - many positive IS 37C IAT I II Drug Binds to RBCs (Hapten) Drug Adsorption Penicillin/penicillin derivatives & cephalosporins Drug binds covalently to membrane proteins and stimulates hapten-dependent antibodies Antibody reacts with normal RBCs pretreated with drug Drug-Dependent Drug Does Not Coat RBCs Immune Complex Quinidine, quinine, NSAIDs Through an unknown mechanism, drug induces antibodies that bind to RBC only when drug is present in soluble form Antibody reacts with RBCs when soluble drug is present Drug-Induced Autoantibody Alpha methyldopa, procainamide Through an unknown mechanism, drug induces autoantibodies specific for RBC membrane proteins Antibody reacts with normal RBCs in the absence of drug Nonimmunologic Protein Adsorption (NIPA) Membrane Modification Cephalosporins Tazobactum, Clavunate Drug coats RBCs and causes them to become sticky DAT - weakly positive Rarely associated with DIIHA Untrtd RBCs Testing Drug-Treated RBCs Patient Serum Pos Con Normal Serum 15 RT C IAT RT C 0 IAT 0 55

20 AIHA Testing in Presence of Drug e+ RBCs 30 RT 60 37C IAT Patient Serum + Drug Patient Serum + Diluent Diluent + Drug Eluate + Drug 0 0 0/3+ Drug-Induced Hemolytic Anemia Treatment STOP the DRUG!!!! Treat the symptoms Don t take drug in the future Eluate + Diluent Positive Control + Drug AIHA & DIIHA - Things to remember Always look for medication history in a question but don t assume it s the culprit Look at strength of reactivity of DAT Correlate DAT reactivity with patients clinical information DAT strongly positive, Eluate negative is key finding in DIIHA BUT patient should be showing clinical signs of hemolysis 22 Case Studies all antibody problems, simple to complex 21 Case Studies all with positive DATs Questions & Answers covering broad range of TM topics Reading List AABB Technical Manual, 18th ed, 2014, Chapter 17, p , Methods, Section 4 (Flash Drive) Petz LD, Garratty G Acquired Immune Hemolytic Anemias Philadelphia: Churchill Livingstone, 2004 Judd WJ, Johnson ST, Storry JR Judd s Methods in Immunohematology, 3 rd ed, p , Section XI, 2008 Lechner K, Jager U How I treat autoimmune hemolytic anemias in adults Blood 2010;116(11): Barcellini W, et al Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients Blood 2014;124(19): Immunohematology 2014;30 (2) Special Edition on Drug-Induced Immune Cytopenias WJ Judd, ST Johnson, JR Storry Judd s Methods in Immunohematology, 3 rd ed 2009 AABB Press Questions? suejohnson@bcwedu 56